Live-Cell Imaging of Long Noncoding RNAs Using Molecular Beacons

  • Yachen Ying
  • Shiqi Mao
  • Christopher J. Krueger
  • Antony K. ChenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)


Long noncoding RNAs (lncRNAs) are a family of non-protein-coding RNA transcripts greater than 200 nucleotides in length that have been regarded as crucial modulators of gene expression in various biological and disease contexts, but mechanisms underlying such regulation still remains largely elusive. In addition to cell lysate-based approaches that have proven invaluable for studies of lncRNAs, live-imaging methods can add value by providing more in-depth information on lncRNA dynamics and localizations at the single-molecule level. Recently, we have developed a versatile imaging approach based on molecular beacons (MBs), which are a class of fluorogenic oligonucleotide-based probes with the capacity to convert RNA target hybridization into a measurable fluorescence signal. In this chapter, we describe the detailed protocol of using MBs to illuminate lncRNA transcripts at the single-molecule level in living cells.

Key words

Molecular beacons lncRNA Tandem repeats Single-molecule 



This project was supported by grants from the National Key R&D Program of China (Nos. 2016YFA0501603 and 2016YFA0100702), the National Natural Science Foundation of China (Nos. 31771583 and 81371613), and the Beijing Natural Science Foundation (7162114).


  1. 1.
    Ulitsky I, Bartel DP (2013) lincRNAs: genomics, evolution, and mechanisms. Cell 154(1):26–46. Scholar
  2. 2.
    Sabin LR, Delas MJ, Hannon GJ (2013) Dogma derailed: the many influences of RNA on the genome. Mol Cell 49(5):783–794. Scholar
  3. 3.
    Quinn JJ, Chang HY (2016) Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet 17(1):47–62. Scholar
  4. 4.
    Holoch D, Moazed D (2015) RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 16(2):71–84. Scholar
  5. 5.
    Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14(3):303–308. Scholar
  6. 6.
    Chen M, Ma Z, Wu X, Mao S, Yang Y, Tan J, Krueger CJ, Chen AK (2017) A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level. Sci Rep 7(1):1550. Scholar
  7. 7.
    Yoon JH, Abdelmohsen K, Kim J, Yang X, Martindale JL, Tominaga-Yamanaka K, White EJ, Orjalo AV, Rinn JL, Kreft SG, Wilson GM, Gorospe M (2013) Scaffold function of long non-coding RNA HOTAIR in protein ubiquitination. Nat Commun 4:2939. Scholar
  8. 8.
    Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HY (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329(5992):689–693. Scholar
  9. 9.
    Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. Scholar
  10. 10.
    van den Bogaard PT, Tyagi S (2009) Using molecular beacons to study dispersal of mRNPs from the gene locus. Methods Mol Biol (Clifton, NJ) 464:91–103. Scholar
  11. 11.
    Zhao D, Yang Y, Qu N, Chen M, Ma Z, Krueger CJ, Behlke MA, Chen AK (2016) Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2'-O-methyl RNA molecular beacons. Biomaterials 100:172–183. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yachen Ying
    • 1
  • Shiqi Mao
    • 1
  • Christopher J. Krueger
    • 1
    • 2
  • Antony K. Chen
    • 1
    Email author
  1. 1.Department of Biomedical EngineeringCollege of Engineering, Peking UniversityBeijingChina
  2. 2.Wallace H Coulter Department of Biomedical EngineeringGeorgia Institute of TechnologyAtlantaUSA

Personalised recommendations